Induction and differentiation of exocrine pancreatic tumors in the rat

Exp. Path. 28,67-87 (1985) Department of Pathology, Northwestern University Medical School, Chicago, Illinois, U.S.A.

Review Induction and differentiation of exocrine pancreatic tumors in the rat By M. S. RAO and J. K. REDDY With 13 figures (Received February 1, 1985)

Address for correspondence: Dr. M. S. RAO, Department of Pathology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611, U.S.A. Key w 0 r ds: pancreatic carcinoma; acinar cell tumor; differentiation; transplantable pancreatic tumor

Introduction Epidemiologic studies indicate a remarkable increase in the incidence of pancreatic carcinoma in the United States and other industrialized nations over the past 3 or 4 decades (FRAUMENI 1975). In the U. S. pancreatic carcinoma ranks fifth in incidence among all cancers, and is the fourth leading cause of cancer death (ACS 1985). Although the reasons for this increasing incidence are not clear, several factors such as high fat diet, smoking, alcohol, diabetes and increased use of coffee have been implicated (WYNDER 1975; MACMAHON 1982; MACK 1982). Human pancreatic carcinoma has been dubbed as a "dismal disease" because of poor prognosis since these cancers grow undetected and when they become clinically apparent have already spread beyond the confines of the pancreas (FITZGERALD 1976). The biological behavior of this neoplasm is poorly understood. Further, there are no biological or immunological markers, at least as of now, to aid in the early diagnosis. Because of the aforementioned reasons, investigations aimed at elucidating the possible etiological factors have increased, as have attempts to develop suitable animal models for study of its histogenesis, biological behavior and identification of tumor associated markers. In an effort to further our understanding of this disease, animal models have been developed in guinea pigs, rats, hamsters and mice using a variety of chemical carcinogens during the past decade. (For reviews see REDDY et aI. 1979; LONGNECKER et aI. 1984a; SCARPELLI et aI. 1984). The animal models, especially rat and hamster, have allowed identification of at least 16 pancreatic carcinogens and advanced our knowledge about the role of pancreas in metabolism of some of these compounds (table 1). It has been shown that pancreas of guinea pigs, hamsters and rats is equipped with necessary mixed function oxidases (MFO) to metabolize inactive carcinogens to active metabolites which react with DNA and other macromolecules. SCARPELLI et aI. (1982) have clearly demonstrated in hamster that all component cells of pancreas are capable of metabolizing N-nitroso-2,6-dimethylmorpholine (NNDM), a pancreatic carcinogen in this species. However, utilizing electron microscope autoradiographic techniques, REZNIK-SCHULLER et aI. (1980) have observed localization of labeled NNDM in acinar und duct cells, but not islet cells. IQBAL et aI. (1977) reported the presence of MFO activity in S-9 fraction and microsomal pellet of pancreas from male strain 13 guinea pigs. The pancreatic MFO are inducible, like in the liver, by methylcholantherene, p-naphthoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin (SCARPELLI et aI. 1980). These various experimental models have also shed considerable light on the histogenesis of pancreatic carcinoma (REDDY et aI. 1979). The cell(s) from which pancreatic tumors arise appear different in different species. Regardless of the type of carcinogen used, in rats the

5*

67

Table 1 Animal models of chemically induced pancreatic carcinoma Species

Carcinogen

Carcinomas Induced

Reference

Guinea Pig

N-methvl- N-nitrosourethane Nvmethyl-Nenitrosourea

Adenocarcinoma Adenocarcinoma

(DRUCKREY et al. 1968) (REDDY and RAO 1975)

Hamster

Disoparpanolnitrosamine Ductal 2,2:-dihydroxy-di-n-propylnitrosamine N-nitrosobis(2-acetoxypropyl)amine Ductal N-nitrosobis(2-oxopropyl)amine Ductal, rare acinar N-nitroso(2-hydroxypropyl) (2-oxopropyl)amine Ductal N-nitrosomethyl(2-oxopropyl)amine Ductal N-nitroso-2,6-dimethylmorpholine Ductal

Rat

(POUR et al. 1974) (POUR et al. 1975) (POUR et a. 1976) (POUR et al. 1977) (POUR et al. 1979) (POUR et al. 1980) (MOHR et al. 1977)

7,12-dimethylbenzanthracene 4-hydroxyaminoquinoline-1-oxide

Acinar Acinar

Azaserine

Acinar

Nafenopin Clofenapate

Acinar Acinar

N-(N-methyl-N-nitrosocarbamoyl)L-ornithine

Acinar

N-nitroso-(2-hydroxypropyl) (2-oxopropyl)

(LONGNECKER et al. 1980)

Acinar

(LONGNECKER et al. 1984b)

(DISSIN et al. 1975) (KONISHI et al. 1976 a, b) (LONGNECKER and CURPHEY 1975) (REDDY and RAO 1977) (SVOBODA and AZARNOFF 1979; REDDY and QURESHI 1979)

pancreatic exocrine carcinomas display acinar cell features whereas in hamsters and the guinea pig the pancreatic tumors are ductlike. The relative role of acinar and ductal epithelium in the histogenesis of pancreatic tumors in different species has generated considerable debate (REDDY et al. 1979; LONGNECKER 1983; FLAKS 1984; POUR 1984; SCARPELLI et al. 1984). In this paper we review different models of pancreatic carcinogenesis in the rat and also consider the usefulness of the transplantable pancreatic tumors in investigating various aspects of differentiation of neoplastic acinar cells.

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A. Spontaneous pancreatic tumors

Spontaneous lesions in the exocrine pancreas of rats are very rare. In a detailedhistological study of the pancreas, ANDREW (1944) found only locule formation (probably corresponding to cystadenomas of current terminology) in 13 of 24 middle aged and old rats. In a postmortem examination of pancreata from 1,252 rats, ROWLATT and ROE (1967) observed exocrine adenomas in 5, adenocarcinomas in 3 and islet cell tumors in 2 animals. In our own study, 2 of the 8 rat that were injected with 0.005N HCI (controls for 4-HAQO experiment) and sacrificed 1 year later showed acidophilic nodules (RAO et al. 1982). In another study ROEBUCK et al. (1984) have reported 4 acidophilic and 9 basophilic foci in 18 control animals. In a recent study, CHIU (1983) reported spontaneously developed hypertrophic foci of acinar cells in 17.6 % of males, 16.8 % of female CD rats ranging from 4-27 months age. He described these lesions as a distinct entity, but considered them to be neither hyperplastic nor neoplastic. Rats that were given corn oil or fed raw soya flour (RSF) also showed increased incidence of pancreatic lesions (MCGUINNESS et al. 1980; BOORMAN and EUSTIC 1984a). When RSF supplemented with minerals and vitamins was fed to rats, pancreatic hyperplastic nodules 68

Exp. Path. 28 (1985) 2

and adenomas developed in 100 % and 80 'Yo respectively by 90 weeks. About 15 % of the rats that were maintained on RSF for more than 90 weeks also developed invasive adenocarcinomas. The control animals maintained on heated soya flour, hyperplastic nodules and adenomas developed in only 45 % and 7 (Yo respectively. No carcinomas were observed. In a study conducted by the National Toxicology Programme it has been found that rats given corn oil as a control vehicle, developed pancreatic acinar cell hyperplasia in 12.6 %, acinar cell adenomas in 4.9 % and acinar carcinoma in 0.27 oft) (BOORMAN 1984 b). From these studies, it is not clear whether RSF and corn oil are acting as carcinogens or promoters of "spontaneously" initiated lesions.

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B. Experimentally induced pancreatic neoplasms

1. 4-Hydroxyaminoquinoline-1-oxide (4-HAQO) model 4-HAQO, the presumed proximate carcinogen of 4-nitroquinoline-l-oxide, is shown to be a pancreatic carcinogen by HAYASHI and HASIGAWA as early as 1971. A single i.v. injection of 4-HAQO in Sprague-Dawley rats at dose levels of 6, 9 and 10 mg/kg body mass induced foci of atypical acini and atypical cell nodules (adenomas) between 162 and 440 days in 100 % of the males and 50 % of the females. These findings were later confirmed by SHINOZCKA and KONISHI (1974) and SHIXOZUKA et al. (1976) who further studied in detail the ultrastructural features of acinar cell lesions. The nodules were small, measured 0.4 to 1.5 mrn and consisted of atypical acinar cells showing irregular nuelei, interdigitating lateral plasma membranes and incompletely formed zymogen granules bearing morphological resemblance to acini of embryonic pancreas. Recently RAO et al. (1982) han studied the effect of a single i.v. injection of 4-HAQO (6 mg/kg b.wt) in male Wistar rats. All animals that were killed between 50 and 54 weeks contained atypical acinar cell foei (.\c\CF) and atypical acinar cell nodules (AACN). (These

Fig. 1. Basophilic focus from a rat treated with a single dose of 4-HAQO. HE

x 210.

Exp, Path. 28 (985) 2

69

Fig. 2. Electr on micrograph of a cell from basophilic focus showing a larger irr egular nucleus and cytoplasm with an abundant amount of RER. X 9,600.

70

Exp, Path. 28 (1985) 2

Fig.!.3A. Acidophilic focus from a 4-HAQO treated rat showing large acini with hyperchromatic nuclei. HE x 210.

Fig.3B. H3-thymidine autoradiograph of acidophilic focus showing many labeled cells (thin arrows). Two cells in mitoses are also present (thick arrows). HE x 310.

Exp. Path. 28 (1985) 2 71

Fig. 4. Electron micrograph~of cells from an acidophilic focus showing large number of electron dense zymogen granules. x 9,GOO.

72 Exp, Path. 28 (19.85) 2

lesions are described as "foci" if they are microscopic in size, and "nodules" if they are grossly visible.) Based on the tinctorial properties and cytoplasmic morphology, such lesions were classified as basophilic foci (BF), acidophilic foci (AF) and acidophilic nodules (AN). The cells in the basophilic foci (fig. 1) were large with irregular nuclei and contained markedly basophilic cytoplasm with few zymogen granules. The cells were arranged as acini and are of the same size as adjacent normal acini. Ultrastructurally, the cells of BF showed interdigitating irregular lateral plasma membranes, scant zymogen granules and a rich complement of parallel arrays of rough endoplasmic reticulum (fig. 2). The cells in AF and AN were also arranged as acini, that were larger than adjacent normal pancreas (fig. 3 a). The nuclei were basally located and the cytoplasm filled with zymogen granules (fig. 4). Electron microscopy confirmed the light microscopic findings, characterized by the cytoplasm filled with electron dense zymogen granules. Parallel rows of RER with thin cisternal spaces were seen scattered in the basal and parabasal areas. In a few cells the RER was dilated and contained variable num bers of intracisternal granules. Both the BF and AF were not encapsulated. In addition to the morphology, significant differences were also observed in the proliferative capacities of cells in AF and BF. In the AF mitotic and labeling indices were 2.7 ± 1.2 and 23.2 ± 3.1(1,000 cells respectively; whereas, in BF the mitotic and labeling indices were 0.1 and 1.2(1,000 cells respectively similar to the adj acent uninvolved pancreas (fig'. 3 b). In the experiments described above which involved injection of a single dose of 4-HAQO in rats the pancreatic lesions are benign. However, slight experimental modification such as administration of 4-HAQO during pancreatic regeneration, may lead to the development of benign as well as malignant pancreatic tumors. KO:"ISHI and his associates (1976 a, b) injected 4-HAQO (7 mg(kg) during maximal DNA synthesis induced after partial pancreatectomy or ethionine induced regeneration. After partial pancreatectomy, hyperplastic nodules, adenomas and adenocarcinomas developed in 86, 93 and 60 % of rats respectively. After ethionine induced regeneration hyperplastic nodules, adenomas and carcinomas developed in 50,92 and 12% respectively; whereas in controls given 4-HAQO during nonregenerative phase only hyperplastic nodules (90 %) and adenomas (30 O~) developed. When 4-HAQO is administered by i.p, or i.v, routes, the effects of this compound appear to be pancreas specific which may be attributable to increased uptake and accumulation in the pancreas. Acute studies have shown that 4-HAQO induces necrosis followed by regeneration of pancreas in both guinea pigs and rats (KO:"ISHI et al. 1974; REDDY et al. 1975). 2. Azaserine-model pancreatic tumors Azaserine (o-diazoacetyl-L-serine), an antimetabolite isolated from cultures of Streptomyces inhibits de novo synthesis of purine (BARTZ et al. 1954; LEVE:"BERG et al. 1957). In rats azaserine is concentrated in the pancreas and kidneys and induces toxic effects in pancreas, kidneys and liver (STER:'fBERG et al. 1957; LOXG:"ECKER and CURPHEY 1975). Azaserine is a mutagen in Ames assay using S. typhimuritlm strains, induces pancreatic DNA damage and inhibits DNA synthesis in the rat pancreas (LO:'fG:"ECKER and CURPHY 1975; LIL.TA et al. 1977; KO:'i'ISHI et al. 1976c). Intraperitoneal injection of azaserine at doses of 5 to 10 mg once or twice a week for 6 months in Wistar rats, and sacrificed 6 to 18 months after initiation of the treatment AACN, adenomas and acinar cell carcinomas developed in 100 %' 28% and 56% of the rats respectively (LOXG:'i'ECKER and CRAWFORD 1974; LONGNECKER and CURPHEY 1975; LONGNECKER 1983). AACN were the earliest lesions to develop and observed as early as 2 months after the beginning of azaserine treatment. AACN were circumscribed and not encapsulated, and contained cells with atypical features and increased mitotic rate. Adenomas are also circumscribed and some of them are encapsulated and contain atypical cells with acinar cell features. However, the cytoplasmic features are not as pleomorphic as in carcinomas. The adenocarcinomas showed well differentiated to anaplastic features with most showing acinar cell differentiation. Some of these carcinomas metastasized to lymph nodes, liver and lung (LoXG:'i'ECKER and CURPHEY 1975). At least 2 of these malignant tumors are established as transplantable tumors in syngenic rats (LONGNECKER Exp, Path. 28 (1985) 2 73

et al. 1979). In addition to pancreatic lesion s, tumors were also observed in kidneys, breast, skin and liv er in azaserine treated rats. Th e dev elopment of adenocarcinoma in pancreas is pre ceded by the development of AACN and ade nomas , signifying that th ese lesion s may serve as precursor lesion s for cancer. The pan creatic tumors were also induced with a single, large i.p. inj ection of azaserine, provided the compo un d wa s given during maximum pancreatic D NA sy nt hesis (KOl.\"ISHI et al. 1980). Aza serin e induced pancr eatic lesion can be promoted by di etary manipulation. ROEBUCK et al. (1981) have given multipl e inj ecti ons of aza serine (10 mg/k g b.wt) and maintained the animals on different high fat diet s for 1 year. One hundred percent of the rats that r eceive d diet cont aining 20 % corn oil dev eloped pancreatic tum ors including invasive carc in oma s and a significant ly in creased number of tumors p er p an creas (more th an 10 per pancr eas). In cont rol gr oup and in rats fed high sat urat ed fat (18 % coconut oil) sh owed a 75 % tumor inciden ce and the number of tumors per pancreas were only 2.4 % . Similarly, the azaserine induced carcinogen esis was also markedly enhanced by feedin g rats with raw soya flour diet (MCGUI NNESS et al. 1982). Tha atypical acinar cell foci induced by azaserine were also of two distinct morphological types and could be classified as AF and BF, simila r to tho se observed in 4-HAQO experiments (ROEBUCK et al. 1984). AF show ed incr eased growth properties and were promoted by 20 % un saturated fat diet. BF were stable, quiescent and not promoted by high fat diet. 3. 7,12-Dimethylbenz(a)anthra c ene (DMBA) induced pancreatic tumors A more direct approach was used by DISSIN et al. (1975) to indu ce pancreatic tumors by impl anting 2-3 mg of crystalline DMBA, an indirect polycyclic hydrocarbon carcinogen, in the head of the pancreas. Between 119 and 363 d after implantati on of DMBA 18 of 24 rats developed malignant pancreati c tumors of which 14 were cell differentiated adenocarcinomas a nd three were spindle cell sa rcomas. Common histological features of the epithelial tumors wer e irregular crowded du ct like struc t ures with little interv enin g st roma. Although by light micr oscopy, the pancreatic tum ors r esembled du ctal ade nocarcinomas, by electron micr oscopy th ey showed features of acina r cells charact erized by a bunda nt RER and occasiona l zym ogen gra nules (BOCKMAN et al. 1976 ). Interestingly , in this m od el mo st of the induc ed tum ors were malignant, and only a single adenoma was record ed. H owever, associated with tumors proliferative pl exif orm t yp e of lesion s (tubular complexes) , considered t o b e precan cerou s changes wer e id entifi ed. Ba sed on ultrastructural and three dim ensional wax r econstru ction st udies, BO CK~fAN et a l. (1978) have proposed that the tubular complex es ar e deriv ed fr om acinar cells. Thi s impression was based on a) id entifi cation of transitional st ages betw een acini and tubules and b) the cells forming tubules were ri ch in RER. Whether the tubular complexes were precursors of ca rcinoma or a reactive pro cess to implantation is not clear since some of the controls al so cont ained these tubular complexes . 4. Mi sce l l an eous model s Clofibrate and its structural anal ogs ca use hepatomegaly, marked proliferation of peroxisome s along with enhanced synthesis of peroxisome associated enzymes, and hepatocellular carcinomas in rats and mice (REDDY et al. 1982 a; REDDY et al, 1982 b). Some of these agents also induce pancreatic tumors. REDDY and RAO (1977 a) reported a 20 % incidence of pancreati c tumors (2 adenomas and 1 m etastasizing acinar cell carcinoma) in male F-344 rats fed naf en opin (0.1 % w/w) in diet for 20-22 m onth s. The aden omas wer e 0.5 to 9 em in diameter and sh owed morphological features similar to those observed in azaserin e-treated rats. The acinar cell carcinoma was large ( ~ 6 cm in diameter) and cont ained ar eas of necrosis. Histological examination of this tumor showed well differentiated to poorly diff erentiated acinar cell carcinomat ous features (fig. 5). Acinar cell nature of the tumor was confir med by electron mi cr oscopic exa mina tion. Th e acinar cell carcinoma metastasized to liver. Thi s tumor wa s successfully transplanted into nude mice and syngenic rats and is ext ensively cha ract eriz ed morphologically, fun ctionally and biochemically (REDDY and RAO 1977; RAO and REDDY 1979).

74 Exp. Path. 28 (1985) 2

Fig. 5.!Acinar cell carcinoma induced in rat pancreas by nafenopin showing vague acinar features and frequent mitoses (arrows). HE x 210.

Fig. 6. Acidophilic nodule from a rat treated with ciprofibrate showing large acini containing many zymogen granules. HE x 200. Ex!'. Path. 28 (1985) 2 75

Fig. 7. Pancreas from a rat treated with eiprofibrate showing acini in different stages of pseudoductule formation. HE x 210.

SVOBODA and AZAR~OFF (1979) found 4 pancreas tumors (3 acinar cell adenomas, 1 acinar cell carcinoma) in a group of 25 rats that were fed clofibrate (0.5 w/w) in diet for 72 to 97 weeks and sacrificed at 129 weeks. In another long term study, RAO et al. (1984) have reported a 20 % incidence of acidophilic nodules (fig. 6) in rats fed ciprofibrate (10 mg/kg b. wt) in diet for 60 weeks. In some of these animals pseudoductules were also seen (fig. 7). The features of AN observed in these experiments resembled those described in 4-HAQO treated rats. N-(N-rnethyl-N-nitrosocarbarnoyl)-L-ornithine (JINCO): :MNCO is a nitrosourea aminoacid which has specific affinity to kidney and pancreas and induces pancreatic DNA damage (LO~GNECKER et al. 1980; LILJA et al. 1978). Long term administration of :MNCO at a concentration of 0.33 mmoles/kg at weekly intervals induced neoplasms in breast, skin, kidney, pancreas and ear duct. The pancreatic lesions included atypical acinar cell nodules in all the animals and adenomas and adenocarcinomas in 3 and 1 of 26 rats respectively. Histological features of these tumors are similar to those induced by azaserine (LONGNECKER and CURPBEY 1975). Nitrosarnines: N-nitrosobis (2-oxopropyl)amine (BOP) and N-nitroso-(2-hydroxypropyl) (2-oxopropyl)amine(HPOP), metabolites of carcinogen di-n-propylnitrosamine, are potent pancreatic carcinogens in hamsters (POUR et al. 1977; POI:R et al. 1983). Both BOP and HPOP were also shown to induce pancreatic DNA damage and tumors in rats (LONGNECKER et al. 1982; LONGNECKER et al. 1984a). Young rats treated with a single dose of BOP (100 mgjkg) or HPOP (20 mgjkg) developed atypical acinar cell nodule within 4 months. Injection of higher dose levels of HPOP (160 mgjkg) resulted in the development of adenomas (79 %), acinar cell carcinomas (71 %)i n addition to AACN at 12 months (LONGNECKER et al. 1984 b). HPOP induced tumors were promoted by 20 % unsaturated fat diet as evidenced by increased incidence and multiplicity. 76

Exp. Path. 28 (1985) 2

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C. Histogenesis of pancreatic tumors in rats

Regardless of the type of carcinogen used, the pancreatic carcinomas that develop in the rat are of acinar cell type with a variable degree of differentiation. In 4-HAQO and azaserine experiments, the development of carcinomas is preceded by the formation of acinar cell foci and nodules showing atypical morphological features, which are regarded as putative precursor lesions. However, the identification of 2 types of foci (AF and BF) raises the question of whether both cell types or only one cell type is preneoplastic. The findings thus far observed indicate that only the cells of AF have enhanced growth potential, whereas cells of BF, albeit stable, do not grow. In these 2 models and peroxisome proliferator treated rats, the precursor lesions and carcinomas display easily identifiable acinar cell features. In DMBAinduced pancreatic carcinoma model the precursor lesions (tubular complexes) resemble duct-like structures and the carcinomas also emulate duct carcinomas. However, both the tubular complexes and carcinomas showed acinar cell features by EM. The duct-like structures are described not only in rats, but also in guinea pigs and hamsters (RAo and REDDY 1980; SCARPELLI and RAO 1978). The change of acinar cells to duct like structures is not peculiar to carcinogen treated animals, and were observed after crush injury (SCARPELLI et al. 1984), implantation of dextran sulfate pellets and during pancreatic regeneration after 4-HAQO induced necrosis (REDDY et al. 1975). The pseudoductule formation first described by REDDY and RAO (1975), may thus represent a putative neoplastic change or a non-specific reaction to a variety of factors. This simple morphologic masquerade of acini as ducts is possible by simple cessation of zymogen production or a defect in packaging of secretory proteins (BOCKMAN et al. 1978). In addition to duct-like structures, the acinar cells or intermediate cells in pancreas can also transform into mature fully differentiated hepatocytes. This phenomenon was observed in hamsters and rats under different experimental conditions (SCARPELLI and RAO 1981; REDDY et al. 1984).

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D. Transplantable pancreatic acinar tumors

1. Origin and morphology Three acinar cell carcinomas of the rat pancreas, one by REDDY and RAO (1977 b) and two by LONGNECKER et al. (1979) have been established as transplantable tumors. These tumors are maintained by serial transplantation in the peritoneal cavity or subcutaneous tissue of either syngeneic hosts or athymio nude mice (RAo and REDDY 1979; LONGNECKER et al. 1979). In addition, RAO et al. (1980) have established in cell culture, a pancreatic acinar carcinoma induced in a rat by azaserine. The transplantable tumor established by REDDY and RAO (1977) was originally induced in an F344 rat fed nafenopin, a peroxisome proliferator. It is a moderately well-differentiated tumor (RAO and REDDY 1979), with cells randomly oriented with respect to one another except in perivascular region where they manifest a polarized pattern with their nuclei oriented toward the vascular basement membrane and the secretory granule-rich cytoplasm oriented in the opposite direction (REDDY et al. 1980; INGBER et al. 1981; IWANIJ et al. 1982b). This tumor exhibits a heterogeneity of cytodifferentiation ranging from cells containing abundant well-developed secretory granules to cells virtually devoid of secretory granules (REDDY et al. 1980; RAo and REDDY 1979). The two transplantable tumors established in Wistar-Lewis rats by LONGNECKER et al. (1979) were induced by azaserine. These two transplantable tumors vary from poorly differentiated to moderately differentiated solid carcinomas with an occasional tendency to form acini. One of these tumors, denoted as CA-20948 (LONGNECKER et al. 1979) with relatively slow growth and showing moderate histologic differentiation has been designated as LONGNECKER pancreatic acinar carcinoma (KANWAR et al. 1984). Variable numbers of secretory granules are present in tumor cells which contain abundant rough endoplasmic reticulum and prominent Golgi complex (fig. 8).

Exp, Path. 28 (1985) 2 77

Fig. 8. Electron micrograph of moderately differentiated transplantable acinar cell carcino ma induce d by azaserine ( LONGN E CKER tumor) showing acinar cells with va riab le numbers of zymogen granules. x 2,500.

78 Exp. Path. 28 (1985) 2

Fig. 9. Electron micrograph of a dissociated acinar cell carcinoma eell from showing abundant zymogen granules. x 5,400.

REDDY

and

RAO

tumor

Fig. 10. Electron micrograph of poorly differcntiatcd carcinoma cell (REDDY and Rxo tumor) lacking zymogen granules. x 6,750.

2. Cell separation The transplantable pancreatic tumors provide an unique opportunity to investigate several interesting facets of neoplastic acinar cell differentiation, morphogenesis and gene expression. The transplantable tumor established by REDDY and RAO (1977) and the LONGNECKER pancreatic acinar carcinoma are easily dissociable into single cells by a technique involving a combination of mechanical fragmentation and EDTA: collagenase dissociation (AMSTERExp. Path. 28 (1985) 2 79

DAM et al. 1978; BECICli and REDDY 1982). The dissociated cell suspension contains> 97 % neoplastic pancreatic acinar cells. The dissociated neoplastic acinar cells, although reveal a continuum of cytodifferentiation (REDDY et al. 1980) two relatively homogenous subpopulations of cells have been isolated by isopyknic Percoll gradient centrifugation (BEClCli and REDDY 1982) from the heterogeneous cell population of the transplantable pancreatic carcinoma established by REDDY and RAo (1977). The subpopulation obtained at a density of 1.0987 glml was designated the granule-enriched fraction and contained morphologically well differentiated cells with abundant mature zymogen granules (fig. 9). The second subpopulation, obtained at a density of 1.0789 glml consisted of poorly differentiated cells lacking zymogen maturation (fig. 10) and is designated granule-deficient fraction. 3. Secretory process and cytodifferentiation By immunocytochemical methods, ten exocrine secretory enzymes have been demonstrated in the secretory granules of well differentiated neoplastic cells (BENDAYAN et al. 1984). Quantitative immunocytochemical method as well as high resolution autoradiographic procedures demonstrated that differentiated acinar carcinoma cells are capable of processing secretory proteins (BENDAYAN et al. 1984; BEClCH et al. 1983). In granule deficient cells, the movement of newly synthesized proteins from RER to the Golgi was noted but it appeared that these cells were unable to concentrate and store them (BECICli et al. 1983). The high resolution autoradiographic studies suggest that the maturation of cytosecretory mechanisms directly related to the state of cytodifferentiation of the neoplastic acinar cell. Since cytosecretory granules of various cell types have been shown to contain sulfated glycoproteins and proteoglycans (REGGIO and PALADE 1978), which are considered to affect the secretory mechanisms of the cell, it was postulated that a defect in sulfation might be responsible for the lack of concentration of secretory proteins in cells with an immature cytodifferentiation (BECICli et al. 1983). A comparison of the rates of [35S] sulfate incorporation in highly differentiated acinar cells of normal pancreas with those of the moderately differentiated cells of the F344 transplantable acinar carcinoma (REDDY and RAo 1977) and the less well differentiated cells of the LONGNECKER transplantable carcinoma showed that the poorly differrentiated cells incorporated more sulfate than did the well differentiated cells (KANWAR et al, 1984). These unexpected results suggest that the inability of secretory granule-deficient cells to store proteins is probably not due to reduced sulfation. Additional studies are needed to determine the extent of sulfation, the reasons for the rapid discharge of newly synthesized proteins and the role of plasmalemmal secretagogue receptors. The cells of the transplantable tumor established by REDDY and RAO (1977) respond to several secretagogues (WARREN et al, 1982; IWANIJ and JAMIESON 1982a) but the maximal discharge from tumor cells was less than that of normal acinar cells. Whether the reduced response is due to cellular heterogeneity or due to secretagogue unresponsive constitutive discharge remains to be determined. The secretagogue response of LONGNECKER pancreatic acinar tumors has not been characterized. 4. Biochemical composition of neoplastic acinar cell secretory granules The homogenates of transplantable pancreatic acinar carcinoma as well as the serum of rats bearing these transplantable tumors had very high levels of amylase and lipase (RAo and REDDY 1979; LONGNECKER et al. 1979). Detailed biochemical analysis of the secretory granules isolated from the pancreatic acinar carcinoma and of the proteins discharged was carried out by HANSEN et al. (1983). HANSEN et al. (1983) analyzed the secretory granules content by two-dimensional polyacrylamide gel electrophoresis to determine whether gene expression in the neoplastic acinar cell is similar to that of normal pancreatic acinar cells. The same 19 proteins found in normal pancreatic secretory granules were also presented in tumor secretory granule extract profiles, except for some quantitative changes in certain basic protein. The significant difference, however, was the presence of a new protein (p 24) with a pI of 5.8 and an apparent Mr 24,000 in the pancreatic acinar carcinoma. The nature and role of this new protein remain to be determined. Of additional importance is that HANSEN

80 Exp. Path. 28 (1985) 2

Fig. 11. REDDY and RAO acinar cell carcinoma maintained on seminiferous tubular basement membrane showing large number of zymogen granules. X 6,000.

6

Exp. Path. 28 (1985) 2 81

et al. (1983) showed that the secretory granule membranes of pancreatic acinar carcinoma of REDDY and RAo (1977) lack the major 80,000 Mr glycoprotein. The reduction or absence of this glycoprotein may account, in part, for the significantly diminished secretory response of neoplastic acinar cells to hormonal stimuli. Additional studies are essential to determine whether the absence of the Mr 80,000 protein in tumor cells is due to alterations in the rates of biosynthesis, glycosylation and possible degradation. A comparison of normal and neoplastic acinar cell biochemical composition and functions should generate new information regarding the role of disordered gene expression in neoplasia. Recently P ARSA and REDDY (1984) investigated the membrane aberrations in chemically induced transplantable rat pancreatic acinar cell adenoma and transplantable pancreatic acinar carcinoma (REDDY and RAo 1977) using a monoclonal murine IgG which detects cellular and intracytoplasmic membranes in normal pancreatic acinar cells. In the acinar cell adenoma the intracytoplasmic membranes, presumably secretory granule membranes stained intensely by indirect immunofluorescence, whereas in pancreatic acinar carcinoma the intracytoplasmic staining was almost completely absent. However, the plasma membranes of pancreatic acinar cell carcinoma cells showed an intensity of staining far exceeding that of normal acinar cells. The significance of the defect in the dynamics of membrane associated antigen(s) in these neoplastic cells and their role in cell proliferation is not known. 5. Modulation of in vitro differentiation In order to investigate certain aspects of cytodifferentiation, modulation of growth, hormone responsiveness and gene expression, establishment of different types of normal as well as neoplastic epithelial cells in culture under defined conditions appears essential. Attempts at maintaining epithelial cells of adult carcinomas have met with limited success. Recently we have utilized rat testicular seminiferous tubular basement membranes (STBM) to maintain in vitro pancreatic acinar carcinoma cells of the tumor established by REDDY and RAo (1977) and LONGNECKER pancreatic acinar carcinoma. Dissociated tumor cells adhered readily to STBM within 1 to 6 h and these STBM-cell aggregates were maintained in conditioned chemically defined medium (PARSAet al. 1970) for up to 7 to 14 d. Two distinct types of differentiation and morphogenetic patterns were noted, the cells of the tumor

Fig. 12. LONGNECKER acinar cell carcinoma maintained on seminiferous tubular basement membrane showing pseudoductular formation. x 120.

82 Exp, Path. 28 (1985) 2

Fig. 13. Electron micrograph of a pseudoductule from LONGNECKER tumor maintained on seminiferous tubular basement membrane showing cells containing variable number of zymogen granules. x 4,000. 6*

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established by REDDY and RAO (1977) formed acinar-like clusters and displayed intercellular junctions and polarization of cytoplasmic organelles (WATANABE et al. 1984). By 4 d, virtually all cells of this acinar carcinoma maintained on STBM showed the characteristics of cytodifferentiation and maturation with numerous secretory granules (fig. 11). In contrast the cells of LONGNECKER pancreatic acinar carcinoma maintained on STBM, reaggregated into duct-like morphogenetic patterns (fig. 12 and 13) and yet appeared to maintain acinar cell function as evidenced by the synthesis of secretory proteins (REDDY et al. in preparation). These duct-like structures resembled tubular complexes observed in pancreas of humans (PARSA et al. 1985) and experimental animals treated with carcinogens (RAO and REDDY 1980; BOCKMAN et al. 1978). Immunofluorescent examination of tubular complexes from human pancreata revealed markers for both acinar and duct cells suggesting ductal metaplasia of acinar cells. The pseudoductular lesions induced in guinea pig by administration of N-methyl-N-nitrosourea (RAo and REDDY 1980) showed features of embryonic pancreatic acinar cells under the electronmicroscope. Similarly the cells lining the tubular complexes induced in rats by DMBA implantation (BOCKMAN et al. 1978) also showed acinar cell features by E.M. examination. These two pancreatic acinar carcinoma cells should prove useful for studies examining the regulation of normal cellular differentiation and morphogenetic controls of neoplastic acinar phenotypes. •

E. Concluding Remarks

The pancreatic tumors that are observed in humans mostly (90 %) display ductal morphology. Similarly, the experimental pancreatic tumors induced in hamster pancreas resemble human adenocarcinomas, although origin from duct cell and dedifferentiated acinar cell is postulated. However, in the rat a variety of carcinogens under different experimental conditions induced only acinar cell lesions. The reason(s) why acinar cells of rat are so sensitive and duct cells so resistent to a carcinogen is not clear. It remains to be examined whether the rat duct cells, like acinar cells, possess various drug metabolizing enzymes. Acknowledgements These studies were supported by NIH Grants CA 23055 and CA 36043.

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